1. Field of the Invention
The present invention relates generally to a method for the detection of DNA sequences. More particularly, the present invention provides a method for the detection of DNA sequences through in situ hybridization and subsequent detection by flow cytometry.
The total length of the DNA helix present in the nucleus of each mammalian cell has been calculated to be about two meters. Under the influence of certain proteins, the DNA of the nucleus is packed into a sphere 5-10 um in diameter. The complex of DNA and protein is called chromatin. Shortly before a cell divides, the DNA becomes even more tightly coiled and is divided into a number of separate, compact packages, which can be observed under the microscope. These are called chromosomes. The number and size of the chromosomes are well-defined for each mammalian species. Human mitotic cells contain 46 chromosomes ranging in size from 2 to 10 .mu.m.
Chromosomes carry the genetic information of a cell. The sequence of the nucleotide bases, which make up the DNA molecule, codes for the required proteins of the species. Each cell of the body contains a complete copy of the DNA and, therefore, a complete set of genetic instructions. The stretches of DNA that code for single hereditary characteristics are called genes. One chromosome may contain thousands of genes. At a given moment, only a small fraction of the genetic material is being translated. The location of the genes on the chromosomes and in relation to regulating sequences is important for their expression in the active genome. Changes in the sequence of the DNA, such as deletions or translocations, may have serious consequences for cell function, and ultimately for the health of the organism. Occasionally, changes in the DNA sequence, and in the protein for which it codes, can be advantageous.
One aspect of the organization of chromosomes of many species can be made visible under the microscope. Special staining techniques produce a striped pattern along the length of the chromosomes. These bands can be seen at high magnification through a microscope. Each chromosome has a characteristic banding pattern. The pattern provides a basis for the identification of the chromosomes. These staining procedures, and more elaborate techniques capable of producing more than 6000 bands in the human genome, have made it possible to detect the structural rearrangements in chromosomes that are reflected in abnormal banding patterns. Chromosomal rearrangements and variations in chromosome number have been associated with various cancers and inheritable human disorders. Radiation, chemical mutagens and viruses have been shown to produce lesions in chromosomes.
Various techniques have made it possible to progress from the study of banding patterns to the study of the molecular organization of chromatin. At a structural level, X-ray diffraction, electron microscopy and antibody labeling are a few of the techniques being used to unravel the 3-dimensional organization of histones, non-histone proteins, and DNA in chromatin and chromosomes.
Knowledge of the organization of chromosomes at the level of the nucleotide sequence itself is increasing at a rapid rate. The sequence, location, and regulation of genes, as well as the protein products of genes, can be studied with the introduction of techniques grouped under the term recombinant DNA technology. These include restriction enzyme digestion, DNA cloning and nucleic acid hybridization. DNA fragments can be multiplied (cloned) by inserting them into plasmids or bacterial viruses, which are then grown in bacteria. An endless variety of DNA sequence probes can be produced in pure quantities in this way. The cloned DNA sequences can also be used to construct a map of the genome in cells and chromosomes.
Using restriction enzymes, the organization of genes in the genome can also be studied. DNA is cut by these enzymes at very specific sites. The resulting DNA fragments are separated by gel electrophoresis. Gene sequence probes are matched by Southern blot hybridization to the highly specific restriction fragment patterns in these gels. For nucleic acid hybridization, the DNA is denatured, or separated into single strands. Under suitable conditions, DNA sequence probes will reanneal with the single stranded DNA to form double-stranded complexes where the nucleotide sequence of the probe and the denatured DNA match well, or are complementary. If the probe is radioactively labeled, the position of the hybridized probe can be visualized by autoradiography. Probes that overlap a splicing point help to link the fragments together in the construction of a complete sequence. In these restriction maps, the molecular organization in normal and abnormal cells of different species can be compared.
Because nucleic acid sequences occupy precise positions in cells and chromosomes, a great deal of information is lost when these molecules are extracted from cells by homogenization. New techniques make it possible to determine the location of specific DNA sequences in chromosomes. In this way, a link can be made between the genomic map and classical cytogenetics. Because chromosomes form a discrete subdivision of the total genome, they can be physically separated. Gene sequence probes can be hybridized in Southern blots or on filters to the DNA isolated from suspensions enriched for individual chromosome types. This is a rapid means of assigning genes to the chromosomes on which they reside.
DNA sequences can be more precisely located in chromosomes using a technique called in situ hybridization. In this technique, the nucleic acid sequence probes are hybridized to chromosomes fixed to slides, after the slide has been exposed to conditions which separate the base pairs in the DNA helix. The specific locations of the bound probe on the arms of the chromosomes can be visualized using radioactive, fluorescent, or enzymatic labeling procedures. The location of the sequences can also be determined in altered chromosomes present in abnormal cells.
Alternatively, purified fractions of a given chromosome can be used as the starting material for the cloning of DNA fragments using recombinant DNA techniques. A set of cloned DNA fragments is called a library. One can obtain, for instance, a library from the X or Y chromosome. Pre-enrichment of a given chromosome before cloning greatly simplifies the search for clones producing DNA sequences unique to that chromosome. "Walking" along the genome, or using overlapping DNA sequence probes to sequentially identify neighboring sequences, is easier when DNA from a single chromosome type has been cloned. Chromosome-specific probes are also useful tools in in situ hybridization studies for the identification of chromosomes, even when they are translocated to a new position, and for the enumeration of chromosomes. Recombinant clones prepared from defective chromosomes can also be a source of probes. These probes can be used to study the defect at a molecular level or to study the disorder's consequences for gene products and cell function.
The foregoing discussion illustrates that there are now many techniques with which chromatin and the genome can be studied. The techniques fall into two general classes: (1) those in which the properties of a population of cells or chromosomes, as a whole, can be characterized; and (2) those that allow the analysis of individual cells or chromosomes, but require their microscopic observation. When the properties of entire populations are averaged, as in the first group of techniques, information about any heterogeneity among the cells or chromosomes is lost. On the other hand, the analysis of individual cells or chromosomes with a microscope is still slow. Without microscope-based microfluorimeters, the observations are difficult to quantify, making them often subjective.
In recent years, flow cytometry instruments have been developed that make it possible to rapidly quantify the optical properties of individual cells, as well as to separate and purify selected cells. The technique of flow cytometry has also been extended to the analysis of chromosomes. If the molecular properties of chromosomes and/or the DNA of the cell nuclei can be successfully translated into optical properties, this technique offers a combination of features that have limited other techniques in the analysis of the genome.
For flow cytometric analysis, cells or cell fragments are suspended in solution and are stained with fluorescent dyes. In the flow cytometer, cells are forced in a narrow stream through the path of an intense light source, such as a laser. The particles pass the laser beam in single file at a rate of several thousand per second. When the cells enter the light spot, they scatter light or emit fluorescence. As each particle passes through the light source, its optical properties are quantified and stored. Large numbers of cells can be measured individually, and the optical properties of a cell population can be determined in a short time. Many flow cytometers also have the ability to sort cells. For this purpose, the sample stream is broken into droplets after the point where the optical measurements are performed. Droplets containing desired cells are given an electric charge and are deflected into a collection tube by the influence of an electrostatic field.
One of the applications of flow cytometry is the measurement of the DNA content of individual cells. During the cell cycle, an accurate copy of the DNA in a cell is made. When a cell divides, the two daughter cells each receive one copy of the DNA. If a population of cells is stained for total DNA content using a DNA-specific fluorchrome, the fluorescence intensity and, thus, the total DNA content, of each cell can be measured in a flow cytometer. Fluorescence distributions, can be measured. The amount of DNA in each cell indicates its position in the cell cycle. Therefore, the distribution of cells among the cell cycle phases can be assessed from the form of the DNA fluorescence distribution. The presence of cells containing an abnormal amount of DNA (which may be due to an extra chromosome or to a chromosome of abnormal size) will also be reflected in the DNA distribution.
At mitosis, the DNA in a nucleus is partitioned over the chromosomes. If the chromosomes are released from mitotic cells, stabilized in solution and stained with fluorescent dyes, they too can be measured in a flow cytometer. Because chromosomes have only a fraction of the DNA content of whole nuclei, the measurement requires equipment with a high degree of sensitivity and precision. The peaks represent individual chromosomes. For this species, all the chromosomes are resolved. The relative peak positions in the distribution contains information about the set of chromosomes in the cells.
Several properties of flow cytometry make it a unique tool for cell biologists. The optical properties of individual cells or chromosomes are analyzed. These properties are expressed in a quantitative and objective manner. High measurement precision allows the detection of small differences in the number of fluorescent molecules bound to each particle in a suspension. Furthermore, the measurements can be performed rapidly. Analysis rates of 1000 events per second are normal, and rates up to 20,000 events per second have been reported. In addition, particles can be sorted from the others in a suspension on the basis of their optical properties for subsequent biochemical or biological assays. Chromosomes, for example, can be purified to be used in gene mapping or cloning experiments.
The application of flow cytometry to the study of cells and chromosomes is constrained by several conditions. To be quantified, a given biological property must be translated into an optical phenomenon, such as fluorescence or scattered light. Thus, a component of interest can be quantified by flow cytometry only when it can be suitably converted into an optical phenomenon. Fluorescent labels and assays have allowed the flow cytometry quantification of such diverse cellular properties as intracellular pH, membrane fluidity, DNA content, protein content, and the presence of membrane antigens. The second constraint is that the cells or chromosomes must exist as individual entities in suspension. This places restrictions on the handling of the particles prior to analysis. Many labeling procedures have been developed for cells or chromosomes fixed firmly to slides. Without this structural support, the cellular components fall apart during many procedures. Thirdly, to obtain relevant biological information from the fluorescence signals of each particle, these signals must be accurately quantified. Variation in the fluorescence intensities of identical particles must be minimized through optimal preparation and staining.
The present application relates to the use of flow cytometry to the study of the structure and molecular organization of the DNA of cell nuclei and chromosomes. The invention provides a method to treat chromatin so that the chromatin can be fluorescently labeled for quantification using flow cytometry. A technique is presented for the stabilization of chromatin in nuclei and chromosomes which allows in situ hybridization to be performed in suspension. A consequence of the method is the detection of specific DNA sequences in interphase nuclei using dual beam cytometry. The properties of the cell that can be analyzed include DNA content, specific DNA sequences base composition and interaction between DNA-specific dyes. Chromosomes derived from clinical material can be analyzed on the basis of these properties.
It would therefore be of great value to provide a method for the detection of DNA sequences nuclei and chromosomes by flow cytometry. For commercial applications, it would be desirable to provide prepared kits containing reagents which are standardized and optimized for sensitivity and accuracy for use in the detection of DNA sequences by flow cytometry.
2. Description of the Prior Art
U.S. Pat. No. 4,358,535 to Falkow et al describes a basic technique for in situ hybridization to determine DNA sequences. In accordance with the method, polynucleotide probes (DNA probes) specific for a DNA sequence (gene) encoding a product characteristic of a pathogen is labeled with a detectable label. A sample suspected of containing a pathogen is transferred onto an inert support, for example, a nitrocellulose filter. The sample is treated in such a way that the cells are localized. The cells are then treated so as to release their DNA and cause it to couple onto the support. Such treatment, as described in the Falkow patent results in the destruction of the cell membrane and the disruption of the nucleus. Subsequent treatment causes a separation of the individual strands of the genome. The strands are then contacted with polynucleotide probes specific for the characteristic polynucleotide sequence under hybridization conditions. Hybridization of the probe to the single stranded polynucleotides from the pathogen is detected by means of the label. The method described in the Falkow patent is suitable for macroscopic examination of DNA sequences but could not be used to examine the DNA sequences by flow cytometry.